EXCEED THE SPACE PROVIDED. The long-term objective is to understand the cellular and molecular mechanisms by which extra- cellular signals alter gene expression. Here we focus on how cellular and molecular mechanisms converge to switch on or off the yeast GAL genes in response to the presence or absence of galactose. Three proteins, Gal3, GalSO and Gal4, provide specificity of galactose sensing and response. Gal4p is a GAL-gene specific DNA-binding gene activator. GalSOp binding to within the Gal4p transcription activation domain, aa 7 6 8- 881 (Gal4pAD), prevents Gal4p-mediated GAL gene expression. In response to galactose and ATP, Gal3p- GalSOp interaction favors dissociation of GalSOp and Gal4pAD. GalSOp also exhibits a two-hybrid interaction with the Gal4p middle region (aa 225-797) (Gal4pMR). Thus, GalSOp has at least two binding surfaces, one for GaISp and one for Gal4pAD, and perhaps another for Gal4pMR. To understand the molecular mechanisms comprising the Gal3, GalSO and Gal4 protein activities, and to lay the foundation for structure/function analyses; 1) GalSOp determinants for its interactions with GaISp and Gal4p, and Gal3p determinants for its interaction with GalSOp will be identified by genetic and physical approaches, 2) genetic and physical methods will be used to determine which protein, GaISp or GalSOp, or both, bind the effector ligands, galactose and ATP, and 3) GaISp, GalSOp, Gal4pAD and Gal4pMR will be purified for protein-ligand and protein-protein analyses and collaborative crystallography. Cellular mechanisms also operate in this system. GalSOp is a nuclear/cytosol shuttling protein and GaISp is predominantly, if not exclusively, restricted to the cytosol. Genetics and microscopy will be used to test the hypothesis that GalSOp shuttling and cytosolic confinement of GaISp are integral to how galactose-triggered GalSp-GalSOp interaction alters Gal80p-Gal4p interaction to switch on GAL genes. The primary goal is to discover how the sub-cellular distribution and dynamics of GaISp, GalSOp and Gal4p are integrated with the relevant ligand-protein and protein-protein interactions to provide proper response to the absence or presence of galactose. The proposed work will elucidate fundamental mechanisms of gene regulation. Insights gained should be broadly applicable; many human genes are regulated by multi-protein switches that are responsive to extra-cellular signals, and it is often such switches that go awry in human disease.